We propose to continue our studies of the molecular mechanisms involved in the replication of bacteriophage Lambda DNA and in the regulation of this process. Our long range goal is to elucidate the biochemical roles of each of the Lambda and E. coli proteins that function in these complex, multienzyme processes. A major focus of our research will be the characterization of the replication of supercoiled Lambdadv plasmid DNA molecules in an in votro system that we have established with a set of purified Lambda and E. coli replication proteins. We will determine the origin and directionality of Lambdadv DNA replication using electron microscopy. We will attempt to define the molecular pathway by which DNA replication is initiated in this system. Isolation and characterization of nucleoprotein structures formed prior to priming will constitute the primary portion of our work in this area. We will use radiolabeled proteins, chemical and nuclease protection studies, as well as immunochemical and electron microscopic techniques to define the morphology and protein components of isolated nucleoprotein replication intermediates. The role of the E. coli dnaG primase in priming DNA synthesis at the Lambda origin will be investigated. The molecular features of the transcriptional activation of Lambda DNA replication in vitro will be determined. This will include biochemical studies aimed at understanding how bacterial histone-like proteins, such as the E. coli HU protein, block the initiation of Lambda DNA replication. Moreover, we will attempt to determine why transcription of the Lambda replication origin by E. coli RNA polymerase counteracts the inhibition mediated by HU protein. Three different replication proteins in this system will receive special attention. We will attempt to thoroughly characterize the physical features of the interaction of the Lambda O replication protein with its recognition site in the Lambda replication origin. We will characterize the interaction of the E. coli dnaB protein with single-stranded DNA and characterize its behavior as it functions as a DNA helicase on long stretches of double-stranded DNA. We will also perform studies aimed at determining the role of the bacterial DNA gyrase during initiation, propagation and termination of Lambdadv DNA replication in vitro. This research will contribute to our understanding of the fundamental molecular processes involved in the initiation, propagation and regulation of DNA replication.